Familial Dysautonomia (FD), also known as “Riley-Day Syndrome” or “hereditary sensory neuropathy type III” (MIM 223900), is an autosomal recessive disorder primarily confined to individuals of Ashkenazi Jewish descent. FD affects the development and survival of sensory, sympathetic, and some parasympathetic neurons (C. M. Riley, et al., “Central Autonomic Dysfunction with Defective Lacrimation”, Pediatrics 3: 468-477 (1949); F. B. Axelrod, et al., Familial Dysautonomia: Diagnosis, Pathogenesis and Management, Adv. Pediatr. 21: 75-96 (1974); F. B. Axelrod, et al., “Familial Dysautonomia”, in: D. Robertson, P. A. Low, R. J. Polinsky (Eds.), Primer on the Autonomic Nervous System, Academic Press, San Diego, pp. 242-249 (1996)) and is caused by mutations in the gene termed IKBKAP which encodes a protein termed IKAP (IκB kinase complex-associated protein) (S. L. Anderson, et al., “Familial Dysautonomia Is Caused by Mutations of the IKAP Gene”, Am. J. Hum. Genet. 68: 753-758 (2001); S. A. Slaugenhaupt, et al., “Tissue-Specific Expression of a Splicing Mutation in the IKBKAP Gene Causes Familial Dysautonomia”, Am. J. Hum. Genet. 68: 598-605 (2001)). IKAP was initially reported to be a scaffold protein involved in the assembly of the IκB kinase complex (L. Cohen, et al., “IKAP is a Scaffold Protein of the IkB Kinase Complex”, Nature 395: 292-297 (1998)), but subsequently was reported to have no association with this complex (D. Krappmann, et al., “The IkB Kinase (IKK) Complex is Tripartite and Contains IKKγ but not IKAP as a Regular Component”, J. Biol. Chem. 275: 29779-29787 (2000)). IKAP is homologous to the Elp1 protein of the Saccharomyces cerevisiae Elongator complex (G. Otero, et al., “Elongator, a Multisubunit Component of a Novel RNA Polymerase II Holoenzyme for Transcriptional Elongation”, Mol. Cell 3: 109-118 (1999)) and is a component of the human Elongator complex (N. A. Hawkes, et al., “Purification and Characterization of the Human Elongator Complex, J. Biol. Chem. 277: 3047-3052 (2002)). IKAP has recently been reported to be a c-Jun N-terminal kinase (JNK)-associated protein capable of JNK stress kinase activation (C. Holmberg, et al., “A Novel Specific Role for 1 Kappa B Kinase Complex-Associated Protein in Cytosolic Stress Signaling”, J. Biol. Chem. 277: 31918-31928 (2002)). The multiple biological activities of IKAP and their roles in FD-mediated neurological deficits remain to be elucidated.
Two FD-causing mutations have been identified in individuals of Ashkenazi Jewish descent. The more common, or major, FD-causing mutation occurs in the donor splice site of intron 20, resulting in aberrant splicing that produces an IKAP transcript lacking exon 20. Translation of this mRNA results in a frameshift that generates a truncated protein lacking all of the amino acids encoded in exons 20-37. The less common, or minor, mutation is a G→C transversion that results in an arginine to proline substitution of amino acid residue 696 of IKAP (S. L. Anderson, et al., “Familial Dysautonomia Is Caused by Mutations of the IKAP Gene”, Am. J. Hum. Genet. 68: 753-758 (2001); S. A. Slaugenhaupt, et al., “Tissue-Specific Expression of a Splicing Mutation in the IKBKAP Gene Causes Familial Dysautonomia”, Am. J. Hum. Genet. 68: 598-605 (2001)).
Mutations that affect RNA splicing are a major cause of human genetic diseases. These diseases may occur as a result of mutations in the splice donor or splice acceptorsequences or in exons or introns, generating cryptic splice junctions. While many of these mutations result in what appears to be an absolute absence of the appropriately spliced gene product, in some cases mutations that affect splicing result in a milder form, or an adult onset form, of the disease in which “leaky” alternative mRNA splicing is observed that produces both mutant (skipped exon) and wild-type (full-length) transcripts (M. L. Huie, et al., “Glycogen Storage Disease Type II: Identification of Four Novel Missense Mutations (D645N, G648S, R672W, R672Q) and Two Insertions/Deletions in the Acid Alpha-Glucosidase Locus of Patients of Differing Phenotype”, Biochem. Biophys. Res. Commun. 244: 921-927 (1998); C. F. Boerkoel, et al., “Leaky Splicing Mutation in the Acid Maltase Gene is Associated with Delayed Onset of Glycogenosis Type II, Am. J. Hum. Genet. 56: 887-897 (1995); S. Beck, et al., “Cystic Fibrosis Patients with the 3272-26A→G Mutation Have Mild Disease, Leaky Alternative mRNA Splicing, and CFTR Protein at the Cell Membrane”, Hum. Mutat. 14: 133-144 (1999); S. Kure, et al., “Glycogen Storage Disease Type Ib Without Neutropenia”, J. Pediatr. 137: 253-256 (2000); I. K. Svenson, et al., “A Second Leaky Splice-Site Mutation in the Spastin Gene”, Am. J. Hum. Genet. 69: 1407-1409 (2001); I. K. Svenson, et al., “Identification and Expression Analysis of Spastin Gene Mutations in Hereditary Spastic Paraplegia”, Am. J. Hum. Genet. 68: 1077-1085 (2001)). The major FD-causing mutation, termed 2507+6T→C or IVS20+6T→C, changes the sequence of the splice donor element of intron 20 from the consensus GTAAGT to a non-consensus GTAAGC, resulting in the generation of a transcript lacking exon 20. This mutation appears to be somewhat leaky as both the mutant and wild-type transcripts are detected in lymphoblasts of individuals homozygous for this FD-causing mutation (S. A. Slaugenhaupt, et al., “Tissue-Specific Expression of a Splicing Mutation in the IKBKAP Gene Causes Familial Dysautonomia”, Am. J. Hum. Genet. 68: 598-605 (2001)).
As FD-derived cells produce the full-length IKAP transcript, it is a goal of the present invention to identify agents that either promote splicing that generates the exon 20-containing transcript or up-regulate IKAP transcription which, due to the somewhat leaky nature of this mutation, could generate increased levels of the correctly spliced transcript and, thereby, more functional IKAP protein in order to treat FD.